Complete mitochondrial genome of the Minois paupera Alphéraky, 1888 (nymphalidae: satyrinae) and its phylogenetic analysis

Abstract The present study firstly reported a complete mitochondrial genome of Minois paupera (Alphéraky, 1888), a Satyrinae species endemic to China. This mitogenome is circular, 15,213 bp in length, and consists of 37 typical mitochondrial genes, including 13 protein-coding genes (PCGs), 22 tRNAs, and two rRNAs. The phylogenetic position was inferred using 31 previously published complete mitogenomes, and the results reveal that M. paupera is the most closely related to Minois dryas. The complete mitogenome of M. paupera provides useful genetic information for further research on the phylogeography and phylogeny of the genus Minois.


Introduction
The genus Minois H€ ubner, which belongs to 'Satyrinae,' known as the mimicry eye spots, is mostly distributed in Palearctic, including one widespread species and several regional endemic species.The genetic relationships of species under this genus are still unclear (Sbordoni et al. 2018).In addition, the unique geographical distribution pattern of this population may be related to historical geological events, which can be used as an excellent research material for biogeography and species differentiation.However, up to now, there are only one complete mitochondrial genome of the dispersed species Minois had been reported, hindering further studies of phylogenetic and phylogeography.
Minois paupera (Alph� eraky, 1888), belongs to Minois and is endemic to China (including in Gansu, Qinghai, Sichuan, N.W. Yunnan, S.E.Tibet) (Lang 2022).The previous studies mainly focused on taxonomy and mostly based on morphological features, lacking the support of molecular evidence.In this study, we sequenced the complete mitochondrial DNA genome of M. paupera to provide baseline data for better understanding its relationship within the genus Minois.

Sample collection and preservation
The specimen was collected from Yongjing County, Gansu Province (103 � 23'32.65"E,36 � 4'5.50"N, 2,160 m).Three legs on the same side were extracted and preserved in ethanol.In terms of morphological characteristics, the dorsal surface yellowish or orange rings around the postdiscal ocelli present on both wings.In addition, male dorsal forewing brand well developed in spaces 1b and 2, and often extending into space 3.The dorsal hindwing postdiscal ocellus in space 2 well developed (Lang 2022).The spread male specimens (Figure 1) were deposited in the Natural History Museum of Sichuan University, Chengdu, China (specimen numbers: SZM742205, contact person: Liang Dou, douliang@scu.edu.cn).

DNA extraction sequencing and genomic assembling
Genomic DNA was extracted from the three legs of a single individual butterfly using the Sangon Rapid Animal Genome DNA Isolation Kit (Shanghai, China).The library preparation and next generation sequencing was finished by Sangon Biotech (Shanghai) Co., Ltd.The libraries were pooled and loaded on Novaseq 6000 (Illumina, San Diego, USA) sequencer by 2 � 150bp paired end sequence kit according to the manufacture's instructions.
Rawbases yielded at least 6 GB were used for downstream analysis.All of the raw reads were trimmed by Fastqc 0.11.2 and assembled the raw sequence reads into contigs by SPAdes 3.15 (Bankevich et al. 2012).The coverage depth is 1x�500x, mean: 190x (Figure S1, Table S1).Finally, complete mitochondrial genome was achieved using the contigs hit against the reference mitochondrial genome as seed sequence through the software MITObim 1.9.1 (Hahn et al. 2013).

Annotation and phylogenetic analysis
The CDS gene boundary was obtained by reverse comparison with the reference genomes of closely related species through NCBI Blastþ 2.28 and GeneWise (Birney et al. 2004).MiTFi (J€ uhling et al. 2012) was used to obtain tRNA sequence annotation, cmsearch (rfam.cm)identifies non-coding RNA by  comparison.Finally, summarizes and collates the complete annotation results.The phylogenetic tree was constructed using PhyloSuite 1.2.3 (Zhang et al. 2020) based on concatenated nucleotide sequences of 13 PCGs and two rRNAs of M. paupera, other 29 representatives from 3 subfamilies of the Nymphalidae, and two outgroup species from Lycaenidae.The nucleotide sequences datasets were initially aligned in batches with MAFFT v7.505 (Katoh and Standley 2013).Ambiguously aligned fragments within the alignments of the 13 PCGs and gap sites within the rRNA sequences were subsequently removed using Gblocks 0.91b (Talavera and Castresana 2007) and trimAl v1.2rev57 (Capella-Guti� errez et al. 2009), respectively.Maximum likelihood (ML) phylogenies were inferred using IQ-TREE v2.2.0 (Nguyen et al. 2015) under the optimal GTR þ F þ R5 model identified by ModelFinder (Kalyaanamoorthy et al. 2017) for 5000 ultrafast (Minh et al. 2013) bootstraps.In addition, CodonW (http:// codonw.sourceforge.net//culong.html)was used to analyze The Relative Synonymous Codon Usage (RSCU).

Phylogenetic position
The ML phylogenetic tree reveals that M. paupera is most closely related to M. dryas, while the genus Minois forms a cluster with Oeneis and Davidina, both supported by 100% values.Furthermore, the monophyly of the genus Minois is well supported in phylogenetic analyses, which aligns with previous phylogenetic studies (Yang et al. 2020;Zhou et al. 2020) and morphological classification research (medium or large size butterflies; antennal club slender, not spatulate as in genus Hipparchia; forewing subcostal vein dilated at base, vein 1b not thickened; hindwing outer margin scalloped, slightly in males, more deeply in females; male upf androconial patch present or absent; forewing two large singlepupilled postdiscal ocelli always present in space 2 andspace 5) (Lang 2022).Notably, Minois species have traditionally been classified within the genus Satyrus, however, there is a dearth of comprehensive molecular data for further validation.Moreover, except for M. dryas, all species in this genus are exclusively found in China and exhibit predominantly regional distributions, but the genetic relationship between these species is still unclear.Therefore, it is imperative to conduct further molecular investigations at the mitochondrial genome level or beyond to elucidate the phylogenetic relationships within this genus (Figure 3).

Conclusions
In the present study, the complete mitogenome of M. paupera was assembled and analyzed.It will provide useful information for improving the taxonomic system of Minois.We found the phylogenetic position of M. paupera within the subfamily of Satyrinae was determined, and the results showed that M. paupera was closely related to M. dryas, and the gene content and arrangement of the newly sequenced mitogenome are similar to those of other determined mitogenomes of Satyrinae.It also provides baseline data for further molecular verification of the biogeographical evolution and phylogenetic relationships within the Satyrinae.

Figure 1 .
Figure 1.The specimen of Minois paupera (Alph� eraky, 1888) used in this study, upperside on the left, underside on the right, scale bar ¼ 10 mm.Photographed and processed by wen-qian Hu.